Diagnostic accuracy of a three-gene Mycobacterium tuberculosis host response cartridge using fingerstick blood for childhood tuberculosis: a multicentre prospective study in low-income and middle-income countries

Summary Background Childhood tuberculosis remains a major cause of morbidity and mortality in part due to missed diagnosis. Diagnostic methods with enhanced sensitivity using easy-to-obtain specimens are needed. We aimed to assess the diagnostic accuracy of the Cepheid Mycobacterium tuberculosis Host Response prototype cartridge (MTB-HR), a candidate test measuring a three-gene transcriptomic signature from fingerstick blood, in children with presumptive tuberculosis disease. Methods RaPaed-TB was a prospective diagnostic accuracy study conducted at four sites in African countries (Malawi, Mozambique, South Africa, and Tanzania) and one site in India. Children younger than 15 years with presumptive pulmonary or extrapulmonary tuberculosis were enrolled between Jan 21, 2019, and June 30, 2021. MTB-HR was performed at baseline and at 1 month in all children and was repeated at 3 months and 6 months in children on tuberculosis treatment. Accuracy was compared with tuberculosis status based on standardised microbiological, radiological, and clinical data. Findings 5313 potentially eligible children were screened, of whom 975 were eligible. 784 children had MTB-HR test results, of whom 639 had a diagnostic classification and were included in the analysis. MTB-HR differentiated children with culture-confirmed tuberculosis from those with unlikely tuberculosis with a sensitivity of 59·8% (95% CI 50·8–68·4). Using any microbiological confirmation (culture, Xpert MTB/RIF Ultra, or both), sensitivity was 41·6% (34·7–48·7), and using a composite clinical reference standard, sensitivity was 29·6% (25·4–34·2). Specificity for all three reference standards was 90·3% (95% CI 85·5–94·0). Performance was similar in different age groups and by malnutrition status. Among children living with HIV, accuracy against the strict reference standard tended to be lower (sensitivity 50·0%, 15·7–84·3) compared with those without HIV (61·0%, 51·6–69·9), although the difference did not reach statistical significance. Combining baseline MTB-HR result with one Ultra result identified 71·2% of children with microbiologically confirmed tuberculosis. Interpretation MTB-HR showed promising diagnostic accuracy for culture-confirmed tuberculosis in this large, geographically diverse, paediatric cohort and hard-to-diagnose subgroups. Funding European and Developing Countries Clinical Trials Partnership, UK Medical Research Council, Swedish International Development Cooperation Agency, Bundesministerium für Bildung und Forschung; German Center for Infection Research (DZIF).


Inclusion criteria
Exclusion criteria 1) Consent and Assent (if applicable): signed written consent/assent, or witnessed oral consent/assent in the case of illiteracy, before undertaking any study-specific activity.
Of the following, either criterion 2), OR criterion 3), or both, has to be met: 2) Confirmation of TB disease: microbiological confirmation of TB disease by positive smear AND/OR culture AND/OR PCR (e.g., Xpert MTB/RIF®); e.g. in a non-study health facility

AND/OR
3) Signs and Symptoms: suspicion of TB disease (one or more criteria): a. Chest X-Ray suggestive of TB: cavity AND/OR hilar/mediastinal lymph node enlarged AND/OR miliary pattern b.Weight loss** or failure to thrive within the previous 3 months that, in the investigator's opinion, is not solely due to inadequate feeding; or to another non-TB cause.c.Any cough combined with: • Loss of weight**; • Evidence of Mycobacterium tuberculosis infection: TST AND/OR IGRA positive d.Cough alone: persistent unremitting cough duration of ≥ 14 days e. Repeated episodes of fever within 14 days not responding to course of antibiotics AND positive TST or IGRA, (for malaria endemic areas: AND after malaria has been excluded by at least a negative rapid test) f.Signs & symptoms of extrapulmonary TB: • Unilateral non-painful lymph node(s) visibly enlarged ≥ 1 month;

Supplementary Table 2: Diagnostic accuracy of the MTB-HR TB score cut-off (1.5) for additional RaPaed-TB cohort subgroups against SRS.
The sample size by subgroup is provided (All), number of true positives (TP), false positives (FP), false negatives (FN), and true negatives (TN), along with the sensitivity, specificity, and area under the curve (AUC) with their respective 95% confidence intervals (CIs).

Supplemental Table 3: Diagnostic accuracy of the MTB-HR TB score cut-off (1•5) for the RaPaed-TB cohort subgroups (age, HIV status, malnutrition, TB severity, TB disease location) against MRS.
The sample size by subgroup is provided (All), number of true positives (TP), false positives (FP), false negatives (FN), and true negatives (TN), along with the sensitivity and specificity with their respective 95% confidence intervals (CIs).
* Denotes missingness ** TB disease severity and location imply that the child already has tuberculosis, hence calculating a specificity was not possible *** The sample size for CD4 category (< 50) was too small and there were no positives.The sample size by subgroup is provided (All), number of true positives (TP), false positives (FP), false negatives (FN), and true negatives (TN), along with the sensitivity and specificity with their respective 95% confidence intervals (Cis).

Subgroups
* Denotes missingness ** TB disease severity and location imply that the child already has tuberculosis, hence calculating a specificity was not possible *** The sample size for CD4 category (< 50) was too small and there were no positives.

Supplemental figures and tables Supplemental Figure 2 :Supplemental Figure 3 :
Assumption tested for one-way repeated ANOVA for MTB-HR TB score distribution over time.A. Assumption of normality tested by the Shapiro Wilk test and by plotting residuals.B and C, assumptions of homoscedasticity tested by plotting the residuals against the predicted values.The MTB-HR TB score was measured at baseline, month 1, month 3, and at month 6 if the child was on treatment or had persisting symptoms.Difference in diagnostic accuracy of the MTB-HR TB score in prospective (A) and biobanked (B) by reference standard.Top.SRS.Middle.MRS.Bottom.CRS A B

A.
SRS. B. MRS. C. CRS Raincloud plots show the distribution of the Ct values for the GBP5 (top), DUSP3 (middle), and KLF2 (bottom) gene transcripts, the bars indicate the 10-90% percentile, and the box the interquartile range (IQR).The points are individual data points and show the spread.The data is displayed stratified by whether the sample was prospective (blue) or biobanked (red) (before recentring).